摘要
牙齿是特殊的矿化器官,由釉质、牙本质、牙骨质和牙髓组成,其中牙本质构成牙齿的主体。牙本质起源于牙源性上皮和外胚间充质的相互作用,进而诱导成牙本质细胞分化成熟,合成和分泌细胞外有机基质,然后羟基磷灰石晶体沉积,矿化开始形成。在矿化过程中,细胞外基质起着非常重要的作用。其中最关键的是牙本质涎蛋白(dentin sialoprotein,DSP)和牙本质磷蛋白(dentin phosphoprotein,DPP),它们来源于同一个编码基因——牙本质涎磷蛋白基因(dentin sialophosphoprotein,DSPP)。长期以来,国内外学者对DSPP基因及其编码蛋白做了大量研究,DSPP基因的表达调控一直是研究的焦点。
近年来,研究人员发现那些过去不被重视的非编码RNA在生物体的生命活动中起着不可忽视的作用,对编码RNA的转录、翻译有着巨大的调控作用,掀起了非编码RNA研究的热潮。1nicroRNA(miRNA)即是非编码RNA大家族中的一贝。miRNAs是一类长约22个核苷酸(nt)的内源性的、非编码的单链RNA分子,它们广泛存在于从植物、线虫到人类的细胞中。迄今为止,在动植物中已经发现了数千个miRNA基因,目前认为在不同物种中,编码miRNA的基因约占蛋白编码基因的1%左右,它们通过与所靶向基因的mRNA的3'UTR区互补结合,实现对靶向mRNA的降解或者翻译抑制,进而作为一类重要的调控分子,参与对生物体的生长、发育、衰老和死亡的调控。在正常生理状态下,生物体内的miRNAs表达呈严格的组织及发育阶段特异性,不同的组织和细胞在发育的不同阶段,miRNA的表达水平有显著差异。在口腔颌面部发育研究领域,Jevnaker等最先运用芯片研究miRNAs在大鼠牙胚和涎腺中的表达,发现在牙胚发育的不同阶段miRNAs表达是动态调控的,一些miRNAs的表达呈组织特异性,并通过生物信息学预测这些miRNAs是牙齿发育,乃至口腔颌面部组织器官发育调控的必需因子。
基于前人的研究成果,为了更全面、深入的探讨miRNA对DSPP表达的调控作用,及其对成牙本质细胞分化的作用,本课题利用生物信息学软件对可能靶向DSPP基因的miRNAs进行预测,并结合双荧光素酶报告基因系统和实时定量PCR的方法筛选和验证能够靶向调控DSPP基因的miRNAs,然后选取其中一个miRNA——mir586做进一步的研究。同样用生物信息学方法预测mir586与DSPP的结合位点,并通过双荧光素酶报告基因系统进行验证。最后,构建过表达mir586和mir586表达抑制的慢病毒表达载体,转染入牙髓细胞,观察在牙髓细胞向成牙本质细胞分化过程中,mir586对细胞功能的影响。
本论文主要包括以下三章内容：
第一章：靶向DSPP基因的microRNAs的筛选和验证
首先利用Targetscan、mirBase、microRNA.org和mirGen-miRanda生物学软件和数据库对可能靶向DSPP基因的miRNAs进行预测,分别得到了17种、15种、10种、10种miRNAs。然后取有两个以上交集的靶位点的miRNA作为研究对象,得到10种miRNAs。为了进一步筛选和验证在成牙本质细胞中能够靶向调节DSPP基因的miRNAs,本研究采用了双荧光素酶报告基因系统和定量PCR的方法。研究结果显示mir885-5p、mir32、mir586能够抑制荧光素酶的活性,即能够对DSPP基因起作用。在牙髓细胞向成牙本质细胞分化过程中,当DSPP基因的表达增多趋于稳定时,mir885-5p、mir32和mir586在10-14 d的表达量都有所下降。结合文献查阅,以及本章所得结果,我们选择抑制荧光素酶活性最强、表达量变化更显著的has-mir586作为进一步研究对象,探讨其与DSPP基因之间的靶向关系,及其对成牙本质细胞分化的作用。
第二章：mir586靶向调节DSPP基因的结合位点分析
首先利用Targetscan和MicroRNA.org软件预测mir586靶向调节DSPP基因的可能结合位点,两者显示了同一结合位点(AAUGCAUG)。为了进一步证实mir586通过此位点对DSPP基因起作用,将结合位点序列突变为UUACGUA,分别构建正常的DSPP 3'UTR表达载体和突变型DSPP 3'UTR表达载体,再通过双荧光素酶报告基因系统,检测荧光素酶活性。结果显示mir586能够与DSPP3'UTR结合,从而抑制荧光素酶活性；而mir586不能与突变型DSPP 3'UTR结合,证明mir586通过与DSPP 3'UTR上190-196碱基序列(AAUGCAUG)结合起作用。同时,进一步证实mir586与DSPP基因以这种“短种子”序列结合,可能起到抑制DSPP mRNA翻译的作用。
第三章：mir586的功能研究
本章主要包括两方面的内容：
1.获取mir586过表达和表达抑制的慢病毒
首先提取正常人全血中的基因组DNA。分别设计mir586过表达序列和表达抑制序列(anti-mir586):从NCBI中查找到has-mir-586的初始序列,选取包含has-mir-586成熟序列及其两端各约200个碱基的序列作为mir586过表达序列；取mir586互补序列进行3次串联,并根据文献设计anti-mir586序列。然后,以提取的正常基因组DNA为模板,PCR扩增mir586基因片段；以anti-mir586聚合引物序列为模板,使之形成双链anti-mir586基因片段。两个基因片段分别与慢病毒表达载体pLVX-shRNA1相连后,转化到感受态DH5α菌中,挑取阳性克隆,提取质粒并进行鉴定。采用Lenti-XTM HTX Packaging System,将构建好的两个慢病毒过表达载体和包装载体Lenti-XTM HTX Packaging Mix,及转染试剂Xfect transfection,转染293T细胞,收集病毒上清,过滤,浓缩,分装,测定病毒滴度约为5×106IFU/ml。最后,慢病毒感染人牙髓细胞,选择合适浓度的G418筛选稳定转染慢病毒的细胞,扩大培养。应用实时荧光定量PCR的方法分别测定mir586过表达、anti-mir586和阴性对照组感染牙髓细胞后的mir586表达情况,结果显示mir586过表达组mir586的表达量比阴性对照组增加了约6倍,而anti-mir586组的表达量减少了约4倍,mir586的表达非常微弱。
2.研究在矿化液诱导牙髓细胞向成牙本质样细胞分化过程中,mir586对细胞增殖和分化的影响
选取矿化液诱导的0、3、7、14和21 d作为测定各项指标的时间点。
采用MTT法评估细胞的增殖情况,发现正常牙髓细胞在矿化诱导分化开始后,随着细胞数目的增多,细胞生长加快,第7 d,细胞逐渐分化、增殖,呈复层生长,矿化结节增多,14 d后细胞密度过高,出现细胞死亡,生长变缓；anti-mir586组细胞在初期时生长较缓慢,第3 d后生长速度较对照组明显加快,到第10 d,生长速度即开始减慢；mir586过表达组细胞数目不断增多,但细胞增长速度较对照组有所减慢。总的来说,在分化过程中,mir586对细胞增殖的影响不大,mir586表达抑制时,可以在一定程度上促进细胞的生长。
通过检测碱性磷酸酶活性评估牙髓细胞的分化情况：正常牙髓细胞矿化诱导下,碱性磷酸酶活性随着分化的进行逐渐增强。mir586过表达组细胞的ALP活性变化不大,后期有上升趋势。而anti-mir586组细胞的ALP活性从第7 d开始显著上升,在矿化诱导14 d达到顶峰。
DMP1和DSPP两个基因编码的蛋白是牙齿发育过程中非常重要的非胶原基质蛋白,被认为是成牙本质细胞的特异性因子。牙髓细胞在矿化液诱导下,可形成成牙本质样细胞,DMP1的表达早于DSPP基因,在诱导分化的第7 d即有大量DMP1表达,14 d达到顶峰,随着分化的进行,DSPP的表达在诱导分化7d后逐渐增多。当mir586过表达时,DSPP的表达速度减慢,表达量减少；DMP1的表达也有所降低。而当mir586受到抑制时,从诱导第7 d开始,DSPP的表达量明显多于对照组；DMP1的表达水平则与对照组没有明显差异。用Western blotting检测DSP蛋白的表达情况时发现：对照组牙髓细胞在矿化诱导第10 d可以看到清晰的DSP蛋白条带,而anti-mir586组在诱导第7 d即可以看到蛋白条带；与对照组相比较,mir586过表达组在诱导第10 d时,蛋白条带明显减弱,表明DSP蛋白被降解,致使表达量减少。
总的来说,mir586表达抑制时,能够促进牙髓细胞向成牙本质样细胞的分化；当mir586过表达时,牙本质特异性基因DSPP、DMP1表达减少,DSP的表达延迟,且表达量明显减少,大大减缓了牙髓细胞分化的进程。
综上所述,本研究通过生物信息学方法预测到多个能作用于牙本质涎磷蛋白基因的miRNAs,通过双荧光素酶报告基因系统和实时荧光定量PCR的方法,重点研究了mir586靶向调控牙本质涎磷蛋白基因的结合位点,并通过构建mir586过表达和表达抑制慢病毒,稳定转染人牙髓细胞,检测矿化液诱导牙髓细胞分化过程中,mir586对细胞增殖和分化的影响。初步研究了miRNAs在成牙本质细胞分化过程中的功能,以及miRNAs对牙本质特异性因子——牙本质涎磷蛋白及其编码基因的调控作用,有利于扩大研究多种miRNAs在牙本质形成中的机制作用,发现新的调控因子和调节途径。
Tooth is a special mineralized organ composed of the enamel, dentin, cementum and pulp, of which dentin is the main part of the teeth. Dentin originated in dental epithelial -mesenchymal interactions, which induced odontoblast differentiation and maturation, the organic synthesis and secretion of extracellular matrix, and hydroxyapatite crystal deposition, mineralization begins to form. In the mineralization process, extracellular matrix plays a very important role. One of the most critical is the dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), which comes from the same gene-dentin sialophosphoprotein gene (DSPP). Over the years, scholars have made a lot of research on DSPP gene and its encoded protein. The regulation of DSPP gene has always been the focus of research.
In recent years, researchers found that those non-coding RNA which were not taken seriously in the past played a significant role in the organisms life activities. They have a huge role in regulating transcription and translation of coding RNA, setting off a wave of non-coding RNA research. microRNA (miRNA) that is a kind of non-coding RNA. miRNAs are a class of about 22 nucleotides (nt) endogenous, non-coding single-stranded RNA molecules, they are widely present in from plants, nematodes to human cells. To date, plants and animals have been found thousands of miRNAs. Currently in different species genes encoded miRNA account for about 1% of genes encoded protein. MiRNAs lead to the targeted mRNA degradation or translation inhibition by targeting mRNA with the 3'UTR, and thus as an important class of regulatory molecules involved in regulating the growth, development, aging and death of the organism. Under normal physiological conditions, expression of miRNAs in vivo have a strict organizational and developmental stage-specific. MiRNA expression of different tissues and cells in different stages of development were significantly different. In the field of oral and maxillofacial development, Jevnaker and colleagues were the first to use microarray studying miRNAs expression in rat teeth and salivary gland, and found miRNAs expression were changing in different stages of tooth development,some of which was tissue-specific. At the same time, bioinformatics analysis suggested these miRNAs were necessary for tooth development and even oral and maxillofacial tissues and organs developmental.
Based on previous research, to study more comprehensivly, in-depth of miRNA regulating DSPP, and its role in odontoblast differentiation, our research uses bioinformatics software to predict miRNAs targeting DSPP gene, combined with dual luciferase reporter gene system and real-time quantitative PCR method to select and validat miRNAs targeting DSPP gene, and then select one of the miRNAs——mir586 to do further research. The same methods are bioinformatics to predict binding sites between mir586 and DSPP and the dual luciferase reporter gene system for validation. Finally, lentiviral vector of mir586 overexpression and mir586 suppression were transfected into human dental pulp cells to discover the effect of mir586 on odontoblast differentiation.
The paper includes the following three parts:
PartⅠ:selection and validation of microRNAs Targeting DSPP gene
Firstly, we use Targetscan, mirBase, microRNA.org and mirGen-miRanda four different bioinformatics software to predict miRNAs targeting DSPP gene, respectively, got 17,15,10,10 miRNAs. Then ten miRNAs were chosen based upon their targeted prediction by more than one program. In order to further select and validat these miRNAs regulating DSPP gene in the odontoblast, the study used a dual luciferase reporter gene system and quantitative PCR methods. The results showed mir885-5p, mir32, mir586 can inhibit the activity of luciferase, which can regulate DSPP gene. During dental pulp cells differentiation to odontoblast, when the DSPP gene expression increased, mir885-5p, mir32 and mir586 expression on day 10-14 have decreased. Combination of literature and the results of this chapter, we chose has-mir586 that strongestly inhibite luciferase activity and express more significantly as a further study to investigate the relationship with DSPP gene, and its effect on odontoblast differentiation.
PartⅡ:analysis of the binding sites between mir586 and DSPP gene
Firstly, Targetscan and MicroRNA.org were used to predict the binding sites between mir586 and DSPP gene. Both show the same binding site (AAUGCAUG). To further confirm the mir586 regulating DSPP gene through this binding site, mutations of the binding site were constructed. Normal DSPP 3 'UTR expression vector and mutant DSPP 3'UTR expression vector were used in the dual luciferase reporter gene system to detect luciferase activity. The results showed that mir586 regulate DSPP 3 'UTR through this binding site, thereby inhibiting the luciferase activity.But mir586 can not combine to mutant DSPP 3' UTR, which proved mir586 combined to sequence of 190～196 of DSPP 3 'UTR (AAUGCAUG).
PartⅢ:the function of mir586
This part mainly includes two aspects:
1. Get mir586 overexpression lentivirus and mir586 inhibition lentivirus.
First, genomic DNA was extracted from normal human whole blood. Sequences were designed for mir586 over-expression and mir586 suppression (anti-mir586).Sequence of has-mir-586 were got from NCBI, and designed mir586 overexpression sequence contains the has-mir-586 mature sequence and 200 base pairs at each end. According to the literature, take mir586 sequence 3 times in series as anti-mir586 sequence. Then, the extracted genomic DNA as template normal, mir586 gene fragment was amplified by PCR. Anti-mir586 primers as a template polymer to make it a double-stranded anti-mir586 gene. Two gene fragments were connected with pLVX-shRNA1 respectively and then transferred into competent DH5 a bacteria. Positive clones were picked out and then plasmids were extracted and testified. With Lenti-X TM HTX Packaging System, two lentiviral vector and packaging vector Lenti-X TM HTX Packaging Mix, and the transfection reagent Xfect transfection were transfected into 293T cells. Then viral supernatant was collected, filtered, concentrate, packaging. Titer of virus were about 5×106 IFU/ml. Finally, lentivirus transfected into human dental pulp cells and use the appropriate concentration of G418 to select stably transfected cells. Real time quantitative PCR method were determined the expression of mir586 among the mir586 overexpression group, anti-mir586 group and negative control group, The result showed that expression of mir586 in mir586 overexpression is as 6 times as that of the negative control group and anti-mir586 group decreased by about 4 times, so expression of mir586 in anti-mir586 group is very weak.
2. During human dental pulp cells cultured in a mineralizing medium differentiation to the odontoblast-like cell, the effect of mir586 on cell proliferation and differentiation
Samples were harvested at 0,3,7,10,14 or 21 days of differentiation to detect indicators.
Cell proliferation was assessed by MTT method and found that the normal dental pulp cells cultured in a mineralizing medium grew quickly with the increase of cells. On day 7, cells gradually differentiation to odontoblast-like cells grew logarithmicly, so that cells were multiple layer growth and mineralized nodules increased. After 14 days the cell density was too high, cell death occurred. The cells of anti-mir586 group grew slowly at the beginning, after 3 days the growth rate were markedly accelerated, to 10 days, the growth rate began to slow down,perhaps because of excessive growth. The number of cells in mir586 overexpression group was increasing,while the growth rate was slower compared to control group. Overall, in the differentiation process, mir586 little effect on cell proliferation. When mir586 expression inhibited, to a certain extent, promote cell growth.
The differentiation of dental pulp cells were evaluated by alkaline phosphatase activity. In normal group, alkaline phosphatase activity were gradually increased with differentiation. ALP activity of cells in mir586 overexpression group changed little, while ALP activity of cells in anti-mir586 group increased significantly from day 7 and to 14 days in mineralization it induced peak.
DMP1 and DSPP proteins encoded by two genes is a very important non-collagen matrix proteins during tooth development, which is considered the odontoblast specific factors. Dental pulp cells cultured in the mineralizing medium can differentiate to odontoblast-like cells. DMP1 gene expresses earlier than DSPP, and with the differentiation DSPP expression increased gradually. From day 7 there were a large number of DMP1 expression and on day 14 it reached peak.When mir586 was overexpressed, DSPP and DMP1 expression were reduced. When mir586 expression was inhibited, DSPP expression was significantly more than the control group from day 7, but DMP1 expression is no significant difference compare to the control group. The expression of DSP was detected by Western blotting. DSP protein bands can be seen clearly in the control group on day 10, which appeared in the anti-mir586 group on day 7.Compared with the control group, protein bands was significantly reduced in mir586 overexpression group from day 10, which indicate that the DSP protein is degraded, resulting in reduced expression.
In general, when mir586 expression is suppressed, mir586 can promote the dental pulp cells differentiating to odontoblast-like cells. When mir586 is overexpressed, the expression of dentin-specific DSPP gene decreased, DSP expression is delayed and decreased, which greatly slowed the process of differentiation.
In summary, this study predicted some miRNAs targeting DSPP by bioinformatics methods. Applying to the dual luciferase reporter gene system and real-time fluorescence quantitative PCR method, we focus on the binding site between mir586 and DSPP. Mir586 overexpression lentivirus and mir586 suppression lentivirus were used to stably transfect to human pulp cells and to detect the effect of mir586 on cell proliferation and differentiation during the differentiation of pulp cells. This is a preliminary study of the miRNAs effect on the process of odontoblast differentiation, and on specific factors of dentin-DSPP and its proteins, which is conducive to extend research of variety of miRNAs in dentinogenesis and to discover new regulatory factors and regulatory pathways.
引文
[1]Veis A, Perry A. The Phosphoprotein of the dentin matrix. Biochemistry. 1967;6:2409-16.
[2]Butler WT, Bhown M, Dimuzio MT, Linde A. Nonocollagenous proteins of dentin. Isolation and partial characterization of rat dentin proteins and proteoglycans using a three-step preparative method. Coll Relat Res. 1981;1:187-99.
[3]MacDougall M, Simmons D, Luan X, Nydegger J, Feng J, Gu TT. Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4. Dentin phosphoprotein DNA sequence determination. J Biol Chem.1997;272:835-42.
[4]Feng JQ, Luan X, Wallace J, Jing D, Ohshima T, Kulkarni AB, D'Souza RN, Kozak CA, MacDougall M. Genomic organization, chromosomal mapping, and promoter analysis of the mouse dentin sialophosphoprotein (Dspp) gene, which codes for both dentin sialoprotein and dentin phosphoprotein. J Biol Chem. 1998;273:9457-64.
[5]Ritchie HH, Wang LH, Knudtson K. A novel rat 523 amino acid phosphophoryn: nucleotide sequence and genomic organization. Biochim Biophys Acta. 2001;1520:212-22.
[6]Gu K, Chang S, Ritchie HH, Clarkson BH, Rutherford RB. Molecular cloning of a human dentin sialophosphoprotein gene. Eur J Oral Sci.2000; 108:35-42.
[7]Yamakoshi Y, Hu JC, Fukae M, et al. Dentin glycoprotein:the protein in the middle of the dentin sialophosphoprotein chimera. J Biol Chem.2005;280: 17472-9.
[8]D'Souza RN, Cavender A, Sunavala G, Alvarez J, Ohshima A,Kulkarni B, et al.. Gene expression patterns of murine dentin matrix protein 1 (Dmpl) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo.J Bone Min Res.1997; 12:2040-9.
[9]Begue-Kirn C, Ruch JV, Ridall AL, Butler WT. Comparative analysis of mouse DSP and DPP expression in odntoblasts, preameloblasts, and experimentally induced odontoblast-like cells. Eur J Oral Sci.1998;106(Suppl 1):254-9.
[10]Qin C, Brunn JC, Cadena E, Ridall A, Tsujigiwa H, Nagatsuka H, Nagai N, Butler WT. The expression of dentin sialoprotein gene in bone. J Dent Res. 2002;81:392-4.
[11]Fisher LW, Jain A, Tayback M, Fedarko NS. Small integrin binding ligand N-linked glycoprotein gene family expression in different cancers. Clin Cancer Res.2004; 10:8501-11.
[12]Chaplet M, Waltregny D, Detry C, Fisher LW, Castronovo V, Bellahcene A. Expression of dentin sialophosphoprotein in human prostate cancer and its correlation with tumor aggressiveness. Int J Cancer.2006; 118:850-6.
[13]Ogbureke KU, Fisher LW. Expression of SIBLINGS and their partner MMPs in salivary glands. J Dent Res.2004;83:664-70.
[14]Ogbureke KU, Fisher LW. Renal expression of SIBLING proteins and their partner matrix metalloproteinases (MMPs). Kidney Int.2005;68:155-66.
[15]Sreenath TL, Cho A, MaeDougall M, Kulkmai AB. Spatial and temporal activity of the dentin sialophosphoprotein gene promoter:differential regulation in odontoblasts and ameloblasts.Int J Dev Biol.1999;43:509-16.
[16]Chen S, Gu TT, Sreenath T, et al. Spatial expression of Cbfal/Runx2 isoforms in teeth and characterization of binding sites in the DSPP gene. Connect Tissue Res. 2002;43:338-44.
[17]Chen S, Rani S, Wu Y, et al. Differential regulation of dentin sialophosphoprotein expression by Runx2 during odontoblast cytodifferentiation. J Biol Chem.2005;280:29717-27.
[18]Chen S, Gluhak-Heinrich J, Wang YH, et al. Runx2, osx, and dspp in tooth development. J Dent Res.2009;88:904-9.
[19]郭婷,曹罡,余擎,等.核心结合因子α 1对牙本质涎磷蛋白基因转录调控的影响.上海口腔医学.2006,25：657-9.
[20]Chen S, Unterbrink A, Kadapakkam S, et al. Regulation of the Cell Type-specific dentin sialophosphoprotein gene expression in mouse odontoblasts by a novel transcription repressor and an activator CCAAT-binding factor. J Biol Chem. 2004;279:42182-91.
[21]Narayanan K, Ramachandran A, Peterson MC, et al. The CCAAT enhancer-binding protein (C/EBP)beta and Nrfl interact to regulate dentin sialophosphoprotein (DSPP) gene expression during odontoblast differentiation. J Biol Chem.2004;279:45423-32.
[22]Narayanan K, Gajjeraman S, Ramachandran A, et al. Dentin matrix protein 1 regulates dentin sialophosphoprotein gene transcription during early odontoblast differentiation. J Biol Chem.2006;281:19064-71.
[23]何文喜,牛忠英,赵守亮,等.转录因子c-Jun和c-Fos在牙本质涎磷蛋白基因表达中的作用.华西口腔医学杂志.2006；24：67-9.
[24]Shiba H, Mouri Y, Komatsuzawa H, et al. Macrophage inflammatory protein-3 alpha and beta-defensin-2 stimulate dentin sialophosphoprotein gene expression in human pulp cells. Biochem Biophys Res Commun.2003;306: 867-71.
[25]Iejima D,Sumita Y,Kagami H, et al. Odontoblast marker gene expression is enhanced by a CC-chemokine family protein MIP-3 alpha in human mesenchymal stem cells. Arch Oral Biol.2007;52:924-31.
[26]Yokose S, Naka T. Lymphocyte enhancer-binding factor 1:an essential factor in odontoblastic differentiation of dental pulp cells enzymatically isolated from rat incisors. J Bone Miner Metab.2010;28:650-8.
[27]Padgett RW, Das P, Krishna S. TGF-beta signaling, Smads, and tumor suppressors. Bioessays.1998;20:382-90.
[28]MacDougall M, Unterbrink A, Carnes D, Rani S, Luan X, Chen S. Utilization of MO6-G3 immortalized odontoblast cells in studies regarding dentinogenesis. Adv Dent Res.2001;15:25-9.
[29]Unterbrink A, O'Sullivan M, Chen S, MacDougall M. TGF beta-1 downregulates DMP-1 and DSPP in odontoblasts. Connect Tissue Res.2002;43:354-8.
[30]Thyagarajan T, Screenath T, Cho A et al. Reduced expression of dentin sialophosphoprotein is associated with dysplastic dentin in mice overexpressing transforming growth factor-β in teeth. J Biol Chem.2001;276:11016-20.
[31]He WX, Niu ZY, Zhao SL. et al. TGF-beta activated Smad signalling leads to a Smad3-mediated down-regulation of DSPP in an odontoblast cell line. Arch Oral Biol.2004;49:911-8.
[32]何文喜,牛忠英,赵守亮,等Smad在牙本质涎磷蛋白基因表达中的作用.口腔医学研究.2005;21：1-3.
[33]高杰,吴补领,何文喜,等TGF-β 1/Smad3信号途径对牙本质涎磷蛋白基因表达的调控.中华老年口腔医学杂志.2008;6：155-82.
[34]高杰,吴补领,何文喜,等.Smad3对小鼠牙本质涎磷蛋白启动子片段的调控.临床口腔医学杂志.2008;24：451-3.
[35]Hwang YC, Hwang IN, Oh WM. et al. Influence of TGF-betal on the expression of BSP, DSP, TGF-betal receptor I and Smad proteins during reparative dentinogenesis. J Mol Histol.2008;39:153-60.
[36]Knight C, Simmons D, Gu TT, Gluhak-Heinrich J, Zeichner-David M, MacDougall M. Cloning, characterization and tissue expression pattern of mouse Nma/BAMBI. J Dent Res.2001;80:1895-1902.
[37]Gonzales CB, Simmons D, Macdougall M. Competing Roles of TGF {beta} and Nma/BAMBI in Odontoblasts. J Dent Res.2010;89:597-602.
[38]Chen S, Gluhak-Heinrich J,Martinez M, et al. Bone morphogenetic protein 2 mediates dentin sialophosphoprotein expression and odontoblast differentiation via NF-Y signaling. J Biol Chem.2008;283:19359-70.
[39]Yamashiro T, Zheng L, Shitaku Y, et al. WntlOa regulates dentin sialophosphoprotein mRNA expression and possibly links odontoblast differentiation and tooth morphogenesis. Differentiation.2007;75:452-62.
[40]Zhang C, Chang J, Sonoyama W, Shi S, Wang CY. Inhibition of human dental pulp stem cell differentiation by Notch signaling. J Dent Res.2008;87:250-5.
[41]He F, Yang Z, Tan Y. et al. Effects of Notch ligand Deltal on the proliferation and differentiation of human dental pulp stem cells in vitro. Arch Oral Biol. 2009;54:216-22.
[42]Butler WT, Bhown M, DiMuzio MT, Cothran WC, Linde A. Multiple forms of rat dentin phosphoproteins. Arch Biochem Biophys.1983;225:178-86.
[43]Lee SL, Veis A, Glonek T. Dentin phosphoprotein:an extracellular calcium-binding protein. Biochemistry.1977;16:2971-9.
[44]Butler WT, Brunn JC, Qin C. Dentin extracellular matrix (ECM) proteins: comparison to bone ECM and contribution to dynamics of dentinogenesis. Connect Tissue Res.2003;44(Suppl 1):171-8.
[45]George A, Bannon L, Sabsay B, Dillon JW, Malone J, Veis A, Jenkins NA, Gilbert DJ, Copeland NG. The carboxyl-terminal domain of phosphophoryn contains unique extended triplet amino acid repeat sequences forming ordered carboxyl-phosphate interaction ridges that may be essential in the biomineralization process. J Biol Chem.1996;271:32869-73.
[46]Ritchie HH, Wang LH. Sequence determination of an extremely acidic rat dentin phosphoprotein. J Biol Chem.1996;271:21695-8.
[47]Ritchie HH, Hou H, Veis A, et al. Cloning and sequence determination of rat dentin sialoprotein, a novel dentin protein. J Biol Chem.1994;269:3698-702.
[48]Qin C, Cook RG, Orkiszewski RS, Butler WT. Identification and characterization of the carboxyl-terminal region of rat dentin sialoprotein. J Biol Chem. 2001;276:904-9.
[49]Qin C, Baba O, Butler WT. Post-translational modifications of sibling proteins and their roles in osteogenesis and dentinogenesis. Crit Rev Oral Biol Med. 2004; 15:126-36.
[50]Yamakoshi Y, Hu JC, Iwata T, Kobayashi K, Fukae M, Simmer JP. Dentin sialophosphoprotein is processed by MMP-2 and MMP-20 in vitro and in vivo. J Biol Chem.2006;281:38235-43.
[51]Steiglitz BM, Ayala M, Narayanan K, George A, Greenspan DS. Bone morphogenetic protein-1/Tolloid-like proteinases process dentin matrix protein-1. J Biol Chem.2004;279:980-6.
[52]Sun Y, Lu Y, Chen S, Prasad M, Wang X, Zhu Q, et al. Key proteolytic cleavage site and full-length form of DSPP. J Dent Res.2010;89:498-503.
[53]Fisher LW, Torchia DA, Fohr B, Young MF, Fedarko NS. Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin. Biochem Biophys Res Commun.2001;19;280:460-5.
[54]Dickson IR, Dimuzio MT, Volpin D, Ananthanarayanan S, Veis A. The extraction of phosphoproteins from bovine dentin. Calcif Tissue Res.1975;19:51-61.
[55]Dimuzio MT, Veis A. The biosynthesis of phosphophoryns and dentin collagen in the continuously erupting rat incisor. J Biol Chem.1978;253:6845-52.
[56]Zanetti M, de Bernard B, Jontell M, Linde A. Ca2+-binding studies of the phosphoprotein from rat-incisor dentine. Eur J Biochem.1981;113:541-5.
[57]Marsh ME. Binding of calcium and phosphate ions to dentin phosphophoryn. Biochemistry.1989;28:346-52.
[58]Stetler-Stevenson WG, Veis A. Type I collagen shows a specific binding affinity for bovine dentin phosphophoryn. Calcif Tissue Int.1986;38:135-41.
[59]Furedi-Milhofer H, Mordian-Oldak J, Weiner S, et al.Interactions of matrix proteins from mineralization tissues with oetaealcium phosphate. Connect Tissue Res.1994;30:251-64.
[60]Lussi A, Crenshaw MA, Linda A, et al. Induction and inhibition of hydroxyapatite formation by dentin phosphoprotein in vitro. Archs Oral Biol. 1988;33:685-91.
[61]Bulter WT. Dentin matrix proteins. Eur J Oral Sci.1998;106(Suppl1):204-10.
[62]MacDougall M, Nydegger J, Gu TT, et al. Developmental regulation of dentin sialophospho protein during ameloblast differentiation:a potential enamel matrix nueleator. Connect Tissue Res.1998;39:25-37.
[63]Saito T, Ogawa M, Hata Y, Bessho K. Acceleration effect of human recombinant bone morphogenetic protein-2 on differentiation of human pulp cells into odontoblasts. J Endod.2004;30:205-8.
[64]Jadlowiec JA, Zhang X, Li J, Campbell PG, Sfeir C. Extracellular matrix-mediated signaling by dentin phosphophoryn involves activation of the Smad pathway independent of bone morphogenetic protein. J Biol Chem. 2006;281:5341-7.
[65]Alvares K, Kanwar YS, Veis A. Expression and potential role of dentin phosphophoryn (DPP) in mouse embryonic tissues involved in epithelial-mesenchymal interactions and branching morphogenesis. Dev Dyn. 2006;235:2980-90.
[66]Wu L, Zhu F, Wu Y, Lin Y, Nie X, Jing W, Qiao J, Liu L, Tang W, Zheng X, Tian W. Dentin sialophosphoprotein-promoted mineralization and expression of odontogenic genes in adipose-derived stromal cells. Cells Tissues Organs. 2008;187:103-12.
[67]D'Souza RN, Bronekers AL, HapPonen RP. Developmental expression of a 53kD dentin sialoprotein in rat tooth organs. J Histochem Cytochem.1992;40: 359-66.
[68]Bronkers AL, D'Souza RN, Butler WT et al. Dentin sialoprotein:biosythesis and developmental appearance in rat tooth germs in comparison with amelogenins, osteoealcin and collagen type Ⅰ. Cell Tissue Res.1993;273:237-47.
[69]Wada Y, et al. Eleetrophoretic gels of dentin matrix proteins as diffusion media for in vitro mineralization. J Dent Res.1996;75:1381-7.
[70]Boskey A, Spevak L, Tan M, Doty SB, Butler WT. Dentin sialoprotein (DSP) has limited effects on in vitro apatite formation and growth. Calcif Tissue Int. 2000;67:472-8.
[71]Suzuki S, Sreenath T, Haruyama N, Honeycutt C, Terse A, Cho A, Kohler T, Muller R, Goldberg M, Kulkarni AB. Dentin sialoprotein and dentin phosphoprotein have distinct roles in dentin mineralization. Matrix Biol. 2009;28:221-9.
[72]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993;75:843-54.
[73]Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs. Science.2001;294:853.
[74]Song L, Tuan RS. MicroRNAs and cell differentiation in mammalian development. Birth Defects ResC Embryo Today.2006;78:140-9.
[75]Lin SL, Chang D, Wu DY, Ying SY. A novel RNA splicing-mediated gene silencing mechanism potential for genome evolution. Biochem Biophys Res Commun.2003;310:754-60.
[76]Lee Y, Kim M, Han J, et al. MicroRNA genes are transcribed by RNA polymerase Ⅱ. EMBO J.2004;23:4051-60.
[77]Lee Y, Ahn C,Han J, et al. The nuclear RNase Ⅲ Drosha initiates microRNA processing. Nature.2003;425:415-9.
[78]Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U. Nuclear export of microRNA precursors. Science.2003;303:95-8.
[79]Yi R, Qin Y, Macara IG, Cullen BR. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev.2003;17:3011-6.
[80]Wu L, Fan J, Belasco J. G, Micro RNAs direct rapid deadeny lation of mRNA. Proc Natl Acad Sci.2006; 103:4034-9.
[81]Kanellopoulou C, Muljo SA, Kung AL, et al. Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev. 2005;19:489-501.
[82]Murchison EP, Partridge JF, Tam OH, et al. Characterization of Dicer-deficient murine embryonic stem cells. Proc Natl Acad Sci USA.2005;102:12135-40.
[83]Houbaviy HB, Murray MF, Sharp PA. Embryonic stem cell-specific MicroRNAs. Dev Cell.2003;5:351.
[84]Tang F, Hajkova P, Barton SC, et al. MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Res.2006;34:e9.
[85]Yang S, Tutton S, Pierce E, et al. Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells. Mol Cell Biol.2001;21:7807-16.
[86]Suh MR, Lee Y, Kim JY, et al. Human embryonic stem cells express a unique set of microRNAs. Dev Biol.2004;270:488-98.
[87]Bernstein E., Kim S. Y., Carmell M. A., et al. Dicer is essential for mouse development. Nat Genet.2003;35:215-7.
[88]Wang Y, Medvid R, Melton C, et al. DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nat Genet. 2007;39:380-5.
[89]Ivey KN, Muth A, Arnold J, et al. MicroRNA regulation of cell lineages in mouse and human embryonic stem cells. Cell Stem Cell.2008;2:219-29.
[90]Tay YM, Tam WL, Ang YS, et al. MicroRNA-134 modulates the differentiation of mouse embryonic stem cells, where it causes post-transcriptional attenuation of Nanog and LRH1. Stem Cells.2008;26:17-29.
[91]Viswanathan SR, Daley GQ, Gregory RI. Selective blockade of microRNA processing by Lin28. Science.2008;320:97-100.
[92]Melton C, Judson RL, Blelloch R. Opposing microRNA families regulate self-renewal in mouse embryonic stem cells. Nature.2010;463:621-6.
[93]Marson A, Levine SS, Cole MF, Frampton GM, Brambrink T, John-stone S, et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell.2008; 134:521-33.
[94]Bae S, Ahn JH, Park CW, et al. Gene and microRNA expression signatures of human mesenchymal stromal cells in comparison to fibroblasts. Cell Tissue Res. 2009;335:565-73.
[95]Esau C, Kang X, Peralta E, et al. MicroRNA-143 regulates adipocyte differentiation. J Biol Chem.2004;279:52361-65.
[96]Kajimoto K, Naraba H, Iwai N. MicroRNA and 3T3-L1 pre-adipocyte differentiation. RNA.2006; 12:1626-32.
[97]Bork S, Horn P, Castoldi M, Hellwig I, Ho AD, Wagner W. Adipogenic differentiation of human mesenchymal stromal cells is down-regulated by microRNA-369-5p and up-regulated by microRNA-371. J Cell Physiol.2010;6.
[98]Lee EK, Lee MJ, Abdelmohsen K, Kim W, Kim MM, Srikantan S, et al. miR-130 suppresses adipogenesis by inhibiting PPARy expression. Mol Cell Biol.2010;6.
[99]Kim SY, Kim AY, Lee HW, Son YH, Lee GY, Lee JW, et al. miR-27a is a negative regulator of adipocyte differentiation via suppressing PPARy expression. Biochem Biophys Res Commun.2010;392:323-28.
[100]Karbiener M, Fischer C, Nowitsch S, Opriessnig P, Papak C, Ailhaud G, et al. microRNA miR-27b impairs human adipocyte differentiation and targets PPARy. Biochem Biophys Res Commun.2009;390:247-51.
[101]Kim YJ, Hwang SJ, Bae YC, Jung JS. MIR-21 regulates adipogenic differentiation through the modulation of TGF-β signaling in mesenchymal stem cells derived from huaman adipose tissue. Stem Cells.2009;27:3093-102.
[102]Wilfred BR, Wang WX, Nelson PT. Energizing miRNA research:A review of the role of miRNAs in lipid metabolism, with a prediction that miR-103/107 regulates human metabolic pathways. Mol Genet Metab.2007;91:209-17.
[103]Mizuno Y, Yagi K, Tokuzawa Y, et al. miR-125b inhibits osteoblastic differentiation by down regulation of cell proliferation. Biochem Biophys Res Commun.2008;368:267-72.
[104]Luzi E, Marini F, Sala SC, et al. Osteogenic differentiation of human adipose tissue derived stem cells ismodulated by themiR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res.2008;23:287-95.
[105]Kim YJ, Bae SW, Yu SS, et al. miR-196a Regulates proliferation and osteogenic differentiation in mesenchymal stem cells derived from human adipose tissue. J Bone Miner Res.2009;24:816-25.
[106]Li Z, Hassan MQ, Volinia S, et al. A microRNA signature for a BMP-2 induced osteoblast lineage commitment program. PNAS.2008; 105:13906-11.
[107]Lin EA, Kong L, Bai XH, Luan Y, Liu CJ. miR-199a, a bone morphogenic protein 2-responsive Micro RNA, regulates chondrogenesis via direct targeting to Smadl. J Biol Chem.2009;284:11326-35.
[108]Tuddenham L, Wheeler G, Ntounia-Fousara S, et al. The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Lett. 2006;580:4214-17.
[109]Goff LA, Boucher S, Ricupero CL, et al. Differentiating human multipotent mesenchymal stromal cells regulate micro RNAs:prediction of microRNA regulation by PDGF during osteogenesis.Exp Hematol.2008;36:1354-69.
[110]Hung PS, Chen FC, Kuang SH, et al. miR-146a induces differentiation of periodontal ligament cells. J Dent Res.2010;89:252-57.
[111]Hornstein E and Shomron N. Canalization of development by microRNA. Nat Genet.2006;38(Suppl):S20-4.
[112]Chen C, Ridzon DA,Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop real-time RT-PCR. Nucleic Acids Research.2005;33:e179.
[113]Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA.2000;97:13625-30.
[114]Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG. SHED:stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci USA.2003; 100: 5807-12.
[115]Alliot-Licht B, Bluteau G, Magne D, et al.. Dexamethasone stimulates differentiation of odontoblast-like cells in human dental pulp cultures. Cell Tissue Res.2005;321:391-400.
[116]Walsh S, Jordan GR, Jefferiss C, et al. High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro:relevance to glucocorticoid-induced osteoporosis. Rheumatology (Oxford).2001;40:74-83.
[117]Pei W, Yoshimine Y, Heersche JN. Identification of dexamethasone-dependent osteoprogenitors in cell populations derived from adult human female bone. Calcif Tissue Int.2003;72:124-34.
[118]Kasperk C, Schneider U, Sommer U, et al. Differential effects of glucocorticoids on human osteoblastic cell metabolism in vitro. Calcif Tissue Int. 1995;57:120-6.
[119]Kasugai S, Shibata S, Suzuki S, et al. Characterization of a system of mineralized-tissue formation by rat dental pulp cells in culture. Arch Oral Biol. 1993;38:769-77.
[120]Kuo MY, Lan WH, Lin SK, et al. Collagen gene expression in human dental pulp cell cultures. Arch Oral Biol.1992;37:945-52.
[121]Couble ML, Farges JC, Bleicher F, et al. Odontoblast differentiation of human dental pulp cells in explants cultures. Calcif Tissue Int.2000;66:129-38.
[122]Ritchie HH, Park H, Liu J, et al. Effects of dexamethasone, vitamin A and vitamin D3 on DSP-PP mRNA expression in rat tooth organ culture. Biochim Biophys Acta.2004; 1679:263-71.
[123]Kitagawa M, Ueda H, Iizuka S, Sakamoto K, Oka H, Kudo Y, et al.. Immortalization and characterization of human dental pulp cells with odontoblastic differentiation. Arch Oral Biol.2007;52:727-31.
[124]Nomiyama K, Kitamura C, Tsujisawa T, Nagayoshi M, Morotomi T, Terashita M, et al.. Effects of lipopolysaccharide on newly established rat dental pulp-derived cell line with odontoblastic properties. J Endod.2007;33:1187-91.
[125]Paakkonen V, Vuoristo JT, Salo T, Tjaderhane L. Comparative gene expression profile analysis between native human odontoblasts and pulp tissue. Int Endod J. 2008;41:117-27.
[126]Santos PS, Hohne J, Schlattmann P, et al.. Assessment of transmission distortion on chromosome 6p in healthy individuals using tagSNPs. Eur J Hum Genet. 2009; 17:1182-9.
[127]Reinhart BJ, Slack FJ, Basson M, et al. The 21 nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature.2000;403:901-6.
[128]Lee I, Ajay SS, Yook JI, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res. 2009;19:1175-83.
[129]Rom UA, Nielsen FC, Lund AH. MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell.2008:;30:460-71.
[130]Tay Y, Zhang J, Thomson AM, et al. Rigoutsos Ⅰ:MicroRNAs to Nanog,Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature. 2008;455:1124-28.
[131]Vella MC, Reinert K, Slack FJ. Architecture of a validated microRNA::target interaction. Chem Biol.2004;11:1619-23.
[132]Rajewsky N. L(ou)sy miRNA targets? Nat Struct Mol Biol.2006;13:754-5.
[133]Didiano D and Hobert O. Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions. Nat Struct Mol Biol.2006; 13:849-51.
[134]Long D, Lee R, Williams P, et al. Potent effect of target structure on microRNA function. Nat Struct Mol Biol.2007; 14:287-94.
[135]Kertesz M, Lovino N, Unnerstall U, et al. The role of site accessibility in microRNA target recognition. Nat Genet.2007;39:1278-84.
[136]Mishra PJ, Humeniuk R, Mishra PJ, et al. A miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance. Proc Natl Acad Sci U S A.2007; 104:13513-8.
[137]Majoros WH and Ohler U. Spatial preferences of microRNA targets in 3' untranslated regions. BMC Genomics.2007;8:152.
[138]Chen CZ, Li L, Lodish HF, et al. MicroRNAs modulate hematopoietic lineage differentiation. Science.2004;303:83-6.
[139]Zeng Y, Cullen BR. Sequence requirements for micro RNA processing and function in human cells. RNA.2003; 9:112-23.
[140]Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges:competitive inhibitors of small RNAs in mammalian cells. Nat Methods.2007;4:721-6.
[141]Singer O, Verma IM. Applications of lentiviral vectors for shRNA delivery and transgenesis. Curr Gene Ther.2008; 8:483-8.
[142]Trowbridge HO. Pulp biology:progress during the past 25 years. Aust Endod J. 2003;29:5-12.
[143]Qin C, Brunn JC, Cadena E, et al. Dentin sialoprotein in bone and dentin sialophosphoprotein gene expressed by osteoblasts. Connect Tissue Res. 2003;44(Suppl):179-83.
[144]Kalajzic I, Braut A, Guo D, et al. Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene. Bone. 2004;35:74-82.
[145]Tsang J, Zhu J, van Oudenaarden A. MicroRNA-mediated feedback and feedforward loops are recurrent network motifs in mammals. Mol Cell. 2007;26:753-67.